Process for preparing vaccines



Patented May 11, 1926.

UNITED STATES PATENT OFFICE.

'rmx momeomn'nv woon, or LOS menus,- canmonnm.

raooEss non ranranme VACCINES.

No Drawing.

My invention relates to detoxicated proteins and bacterial vaccines made from pathogenic micro-organisms and toxic prctelns, generally, and, especially, for use in the treatment of human diseases caused by such pathogenic micro-or anisms and toxic pro-' teins; as well as to t e preparation of such products, including the detoxication of toxic proteins and pathogenic m1cro-organ1sms.

It is Well known that a great many diseases are caused by the presence of toxic proteins in the system, and these are frequently the products of the life processes of pathogenic micro-organisms present therein; and my invention has for its mam object the detoxi cation of such organ sms and substances whereby they may be used in the treatment of such diseases.

1. The exact treatment to which I subject the different materials varies, due to variation in the character and thickness of the capsule and to the variation in the character and content of the toxins produced by different bacteria; but generally stated, I

" treat .the cultures of micro-organisms, or solid media, or of magma, after having been.

centrifuged out of the liquid media with a solution of sodium hydroxide made by mixing -25% of a solution of sodium hydroxide in sterile distilled water, with -75% of additional sterile distilled water,

and this treatment is continued for such .a

length of time (and this varies consider-. ably with the different proteins and micro acetone; the temperatures at which such treatments take place varying from 4 to 100 0., as may be necessary to break the capsule or outer envelope of the micro-or-..

ganism's or substances treated.

- 2. The suspension is then removed to a sterileglass container and treated with pure powdered trypsin 25%, dissolved in sterile distilled water previously made alkaline with Application filed iebruary 29, 1924. Serial No. 696,088. I

1% sodium bicarbonate, and the ferment is allowed to act at 37.5 C. for about five to eight minutes, the time'varying according to v the amount of toxins present in the material treated which areto be rendered inert. In

case the micro-organisms under treatment are capsulated and are. known to produce split proteins during their growth, in the media, I add sufficient sterile distilled water after the trypsin has acted for 2.5 minutes .to assist in carrying the ferment-through the capsule by osmosis and digest the toxic proteins both within and without the capsule. The tone of this solution isthen made normal and this requires sufficient distilled water to balance the bacterial protein, the sodium hydroxide, and the trypsin present,

- whereupon the action of the ferment is continued for 2.5 minutes to completely digest the toxic proteins in and beneath the capsule in capsulated micro-organisms.

3. -I then treat the resulting solution with a 15% solution of sodium oleate in warm sterile distilled water, agitating with a pipette for about 3 minutes to. perfect the soapy solution.

4. I then add 0.5% of phenol and agitate with a pipette to complete solution of the phenol, and then permit it to stand for about 5 minutes.

5.. I then remove the capsule or ectosarc, taking careto remove aslittle of the bacteria as possible, and I do this with the-use of a centrifuge at 1000 revolutions per minute for 1-2. minutes, examining the tube against daylight at say the end of a minute in order to determine whether or not this centrifu ing treatment should be continued, as I o not wish to shake the bacteria completely out of the suspension but only the capsule. The supernatant fluid which contains the pure bacterial protein now divested of its capsule is then poured. If the bacteria is intact, as

determined by microscopical examination, I

standardize this bactenal emulsion by the blood counting method; but in case; the bacteria have been completely split by lysis, I

standardize by comparison with bacterial suspensions of known bacterial content. The antigenic strength of the bacterin may be tested by a complement fixation test and I by animal and therapeutic inoculation to determine its value for antibody production. The addition of sterile distilled water'will now generally be necessary to bring the Q solution nearly to the tone of normal saline solution, and the amount of this addition will be indicated by the total amount of bacterial substance in grams in the original cultural magma, plus the amount of chemi cals and ferment used, and the amount of water added during the process of manipulation. I do this by adding equal parts of sterile distilled water and glycerine to prevent proteolysis and mould contamination, and the solution is rendered normal by hydrometer tests.

Should the alkali be excessive it is reduced by the addition of a solution of 6% acetic acid to the point of protein suspension (a little less than neutral) and the normal tone is then again restored by the addition of sterile distilled water. The bacterin or protein is then tested for sterility by culture and animal inoculation in order to make sure of the absence of all pathogenic microorganisms. (B. Tetani, B. Aerogenes capsulatus, etc.)

The foregoing is a more or less general description of my invention, and I will now give more detailed descriptions as applied to specific bacteria and proteins.

Treatment of cultures of pneumococci and capaulated streptococci, for making detowicated bacterial vaccines and proteins.

1. I treat slant tube cultures or bacterial moist magma of such micro-organisms with sodium hydroxide in the proportions of 0.35 grams to 0.45 grams of bacterial substances with 0.1 cc. of concentrated sodium hydroxide dissolved in 0.14 cc. of sterile, warm, distilled water (a 25% solution of NaOH) by inserting such solution with a pipette into direct contact with the culture of magma, which is then treated for about 5 minutes at 375 (1, preferably in an incubator; and the tone of this solution is approximately normal, since the water content and thev bacterial content with the alkali added approximate 0.85%.

' 2. The action of the sodium hydroxide renders the capsule pervious after such a treatment of 5 minutes, and I then insert by means of a pipette 0.5 cc. of a solution of trypsin ferment which is sterile and chemically pure, made of dissolved 0.25 grams of C. P.'powdered trypsin in 100 cc.

of sterile distilled water, previously made alkaline with 1% sodium bicarbonate and warmed to blood heat; and the trypsin is allowedto act on the culture for 5 minutes in all, but not more. At the end of 2.5 minthis toxinportion is oxidized and digested complete osmosis of the inner bacterial tox- I ins and outer solutions, so that the tone of the two solutions is the same, and it is during this period that the ferment acts on these toxins located in and beneath the capsule, so that these are also rendered inert by the process of digestion. The solution now contains all the products of the bac terial cells; the broken capsules, the inner bacterial protein intact, and the toxic proteins rendered as nearly inert as possible by normal digestion in vitro; and I prefer to stop digestion at this point and maintain the non-toxic inner protein of the microorganisms intact by separating it from the capsular and toxic portions.

3. The next step is the separation of the capsule which is accomplished by treating the solution with 0.3 grams of the 15% solution of sterile sodiunroleate dissolved in warm sterile distilled water, and this is enough to make a total of 30 cc., but the solution becomes normal since the capsule is shaken out. The oleate unites with and fiocculates with the capsule and surrounds the inner bacterial protein with the membrane or film pipette to complete solution thereof. The

solution now contains bacterial magma 0.35 cc. sodium hydroxide in water 0.4 cc., trypsin solution 0.5 cc., water 2. cc., sodium oleate 0.3 cc., phenol 0.5 cc.; making a total of 4.05 cc., and to this I add 4.8 cc. of sterile distilled water to make the solution approximatel normal.

5. then centrifuge the solution thus obtained at 1000 revolutions per minute for 2 minutes, and then examine to ascertain whether the capsule has been shaken out, and hold the'tube against the light to determine if the bacterial suspension is intact. The usual'time for complete separation of the capsule is 5 minutes at. low speed.

6. I then standardize by the blood counting pipette methodor by comparison with known scale solutions of determined bac terial' content, and then add: glycerinc, sodium chloride, and distilled water to normal tone as indicated 'byhydrometer tests. I then examine for pathogenic micro-orthen removed from the media containers and apply'the customary label thereto.

Treatment of non-capswlated staphylococci and streptococci. j

1. 0.1 cc. of concentrated sodium hydroxide in 0.3 cc. of sterile distilled water,

as previously described, is inserted in a culture or'bacte'rial magma and allowed to remain in contact therewith for 3 minutes at 375 C.; or if on the media, until the growth begins to separate from the media. At this point the solution is agitated with the pipette until completely separated and to a sterile glass container.

2. I, now add 0.5 cc. of the trypsin ferment solution previously described and allow this to act for 3 minutes at 37. 5 (1, preferably in an incubator, and I then add 2 cc. of sterile distilled water and allow this to stand 1 minute to complete normal tone.

3. I next add 0.2 cc. of 15% solution of sodium oleate, as previously de'scribed,'and

allow this to stand 1 minute in an incubator,

whereupon it is agitated with the pipette to complete soapy solution for 1 minute. 0.5% of 95% phenol is added and agitated with the pipette to complete solution.

as 4, I then add sufiicient sterile distilled water to approximate normal solution and centrifuge out the capsule, as above described; whereupon I standardize and test for sterility and dilute with equal parts of glycerine and water and sodium chloride to make the solution normal, as set forthabove.

Treatment of Gonococci and Meningeococci.

The treatment of these two cocci is the same and I will therefore describe the treatment of only one, namely the gonocooci.

1. I treat the gonococcus culture or magma with sodium hydroxide the same as I-treat the staphylococci as described above, but I allow the sodium hydroxide solutiont'o act for 5 minutes because these cocci have a thicker capsule.

' described above, allowing minute after the ferment has' acted.

3.4. I then add phenol as described above,

but use no sodium oleate. I then shake-out the capsule, standardize, test for sterility,- etc., as described above. Q" a Treatment of Micrococci 1. I. treat these organisms with hydroxide and trypsin the same as in the case of staphylococci, except that the trypsinx' the treatment of staphylococci.

is added at the same time as the sodium hydroxide and both are allowed to act simultaneously for 5 minutes.

'2, No water is added. 3. Sodium oleate solution is used as in 4. I then treat with phenol as in the case of staphylococci and let stand for an hour, and then agitate with the pipette to homogeneous solution.

5. I then shake out the capsule and proceed as indicatedabove.

Treatment of asthma 1. The culture on' the media is treated as described above for from 5-8 minutes until the growth separates from the media. The balance of the process is the same as that for micrococcus catarrhalis, except that 1% phenol is used on the emulsion for 24 hours,

to secure attenuation. This does not completely devitalize the organism but does. so sufficiently for its use as an antigen -in treating asthma,

2. This organism produces acid and the emulsion must therefore be rendered faintly alkaline to litmus, and this is done by adding strong sodium hydroxide. diluted and standardized and rendered of normal tone by the use of sodium chloride,

It is then act for 2 minutes, whereupon 2 cc. of sterile distilled water are added, and the whole let stand for 1 minute.

3. Add sodium oleate immediately and.

agitate, asdescribed above. 1

4. Shake out the capsule, standardize and render normal and then test/for sterility,

as described above; and then test the toxicity and-the antibody generating power by minute doses, such, for example, as 5 to 50 nillion organisms inhuman beings. Treatment of B. Diphtheria, paeudodiphthcr 'a, acne, baez'lhw of H odghins disease, and other Jiphtherm'd bacilli.

1. Treat with sodium hydroxide solution the same as for staphylococcus.

2;Treat likewise with trypsin. tit-Treat likewise with sodium oleate, in-- asmuch asthese bacilli are autolyzed by the action of sodium oleate.

4. I. standardize only approximately by comparison with bacterial emulsions of the I no same organisms of known bacterial content.

5. Shake out capsules, dilute to normal, and test for sterility, etc. as indicated a ve in the treatment of staphylococci.

Treatment of B. Aerogenes capsulatus of Welek; B. Perez; B. F riedlander; and other bacilli having thick capsules; also B. Amyloclastious z'ntestz'nalz's of diabetes.

1. Treat with a strong sodium hydroxide solution described above long enough to break the capsule, the time varying from 5 to 10 minutes with the different bacilli, this being 5 minutes in the case of the bacilli of diabetes.

' 2. Treat with the ferment simultaneously in the strength above indicated, but insert the ferment 3 minutes after the alkali has acted. and add the sterile distilled water 2.5 minutes after the ferment has acted.

3. Add the sodium oleate and treat as indicated above with respect to staphylococcus.

4. Sterilize with phenol as indicated above and let stand for 2 minutes.

5...Shake out capsules, standardize and bring to normal and determine the sterility,

' etc., as indicated above.

Process for culturing B. Amyloclasticus intestinalis in order to make a detoa'i'eated oat-cine therefrom.

Three methods of culture may be employed to advantage:

1 Shake out the bacillus from patients urine, after incubating same in this urine for 12- hours 'at normal temperature.

2. Culture from the feces on Loefliers liloocll1 serum or plain agar flooded with starch rot 3. Culture from urine or blood on agar flooded with starch broth. The first method may be the method of choice for rapidly obtaining the bacillus for the first vaccine,

and the second and third methods for isola-' tion and corroboration and preparing a stock vaccine.

Process for making a vaccine from baccr'llus amyloclastz'cus intestinalis.

flask for 12 hours at 375 C. in incubator. The presence of the bacillus is indicated by a general clouding of the urine at this time and determined by observation in hanging drop preparation. The presence of a rod shaped bacillus, which grows only on starch containing media with the production of sugar in pure culture is required.

2. Now prepare an emulsion of this bacillus by shaking it out, employing for this purpose a lar 0 tube centrifuge at 2500 to 3500 R. P. for 5 minutes. Determine that the organisms have been completely shaken out by holding the tube against the light, if not, continue at high speed for another three minutes, until the. urine is clear.

3. Now pour off urine as completely as possible without disturbing sediment or loss of bacilli. Suspend sediment in sterile, normal saline solution by agitation with sterile pipette. Then shake-down sediment twice, each time pouring ofi' fluid so as to completely eliminate urine from the solution. At the last treatment cut down the speed of the centrifuge to 1000 R. P. M. and continue only until urinary sediment and cells are thrown down, but not the bacilli. This will require usually 2 minutes Then determine that the bacilli are still in suspension by microscopical examination, then pour off the suspension, which must be free of urinary sediment and cells; determine again by microscopical examination and further centrifuging, if necessary, to secure a perfect suspension of the bacilli only.

4. Treat bacillary magma secured by centrifuging this suspension at high speed for 5 minutes by the method indicated above under the process for the preparation of a detoxicated vaccine from that vacillus. Add 25% of glycerine to concentrated suspension of stock vaccine to prevent proteolysis, when vaccine is to be kept over long periods. This vaccine may be standardized so that 0.5 cc. of the emulsion contains 250,000,000 organisms by dilution with normal saline solution at the time of its use. I

5. The best solid media for culturing the bacillus in pure culture is agar streaked with the patients blood and flooded with sterile broth made from sterilized oatmeal, corn, or rice meals. This broth is made by first obtaining a clear, sterile gruel of these meals, cooking them in sterile water, straining through gauze and sterilization in the Arnold, then diluting them to the consistency of broth with sterile warm water. Plain agar is first streaked with human blood and then inoculated with portions of feces or mucus from the same obtained from a known case of diabetes mellitus, and then flooded with the starch broth 0.5 co. in each tube. Oatmeal and cornmeal broths give equally good results.

6. Pure cultures from the urine and blood may be obtained by adding onecc. of the suspected urine or blood to these agar slants flooded as above with starch broth.

Treatment of B. Tuberculosis for preparing detoacieated tuberculin.

1. Treat sputum containing tubercle bacilli after the method of separating this bacillus by the use of antiformine and shaking to produce homogeneous solution and sedimentation for 12 hours at room temperature in a closed container, and then pour oil the supernatant fluid. The sediment is saline solution and centrifuged.

2. Each 0.35 grams of the bacillus substance so obtained is treated with O.35-milligrams of sodium hydroxide in 1.2 cc. of sterile distilled water for 7-minutes, or as an alternative the bacillary substance may be "treated with 10% sodium hydroxide solution for 12 hours in an incubator at 37 .5 C. until a grayish precipitate is obtained. The excess of fat is removed by extraction of acetone at iii-68 C. for 12-14 hours, whereby *fat-free bacilli are obtained. 3. The defatted bacilli are treated with trypsin 10% (7 in {10 ofidistilled water) using 0.5 cc. of this solution for 15 minutes,

, whereupon 0.55, cc. of 95% phenol are added and agitated to complete solution.

4. The emulsion so obtained is neutralized by the use of 6% acetic acid up to the point of slight alkalinity to litmus. If the neutral or acid point is passed in this neutralization, bacillary substances will precipitate and a suflicient sodium hydroxide solution mustbe added to bring them back into solution. 6.7 cc. of sterile distilled waterds then added, since bacillary substances and chemicals' added amount to 3.8 cc. and thus the solution is brought to normal.

5. Add to the resulting solution 25 cc. of glycerin and sufiicient sterile distilled water to bring the whole up tubercle bacillary substance thus treatedis "diluted with 25 cc. of glycerine and sterile 1. Treat pollen of plants toxic for human beings-in the proportion of 1 gram of pollen substance to 500 cc. of 8.5% (hypertonic sodium chloride solution made up with sterile Berkfield filtered water), b rubbin up the pollen with mortar and pest e' or mac e macerator, adding from time to time. a few 1 drops of glacial acetic acid up to 6% of the total volume or (3 in 50) for one hour,

at which time the outer capsule of the pollen willbe broken and the inner portion of pollen protein released and with the toxins released into the solution.

2. Neutralize the acidity ofthis solution to 100 cc. and then' test for sterility, etc. Each 0.35 grams of by adding suflicient normal sodium hy-' droxide solution, using the drop method until the solution is completely normal to litmus.

3. Filter the solution through sterile cot-' ton, to remove excess. of pollen capsule. Treat the sediment on the cotton again with sodium hydroxide and pass the filtered solution first obtained again through the cotton,

so as to obtain a maximum of pollen sub l? stance.

4. Treat the whole solution thus obtainedwith 1% of trypsin to the total volume of the solution for one hour at 40 C., so as to completely digest andneutralize toxins present. Filter again through sterile filter paper.

5. Add a solution of sodium oleate 15% to 0.5% of the total volume of the solution. This makes a soapy solution. Allow to stand in the incubator until floccules form. This carries down more capsules and toxins, leaving approximately only detoxicated protein of the pollen in solution, which is usedto generate antibodies in susceptible human beings.

6. Add 1 cc. of 95% phenol to each 10 cc. of this pollen extract, shake well and filter through a Berkfield filter. This removes any micro-organisms. Incubate again for 16 hours at 37.5" C. to allow spores of the vegetative organisms to develop. Filter again through the Berkfield filter and culture or test for B. Tetani, gas bacillus or other pathogens by animal inoculation.

- 7. This gives a pollen extract, which contains 1/10,000 milligrams of approximately detoxicated pollen protein to each 0.1 gram .(3 minims) of the solution, .the average dose in adults. Glyoerine in the proportion of 25% may be added to preserve stock solutions. The above process may be repeated in detoxicating proteins which are toxic for humans, varying the length of time of applying the acetic acid and the ferment, ac-

cording to whether the proteins treated are confined in a membrane or not." Toxic proteins free in solution are rendered inert by trypsin action and freedom ,from toxins proven by minimal inoculations.

I claim i proteins and bacterial vaccines which comprises treating bacteria and bacterial toxic proteins withtry sin.

I 2. The process or preparing detoxicated 1. The process for preparing detoxicated proteins and bacterial vaccines which cjomprises treating capsulated bacteria and bacterial toxic protems to make the. capsules pervious and then treating w th trypsm.

3.. 'The process for preparing detoxicated proteins and bacterial vaccmesiwhachcomprises treating capsulated bacteria. and bacterial toxic proteins with to break the p pure powdere capsules and then digesting w'th trypsin.

4. The proccess for preparing detoxicated proteins and bacterial vaccines which comprises treating capsulated bacteria and bacterial toxic proteins with sodium hydroxide to break the ca sules, and then treating with trypsin 25% in a slight alaline solution.

5. The process for preparing detoxicated proteins and bacterial vaccines which comprises treating capsulated bacterial and bacterial toxic proteins to make the capsules pervious and then treating with a. ferment and carrying the ferment through the capsulesb osmosis;

6. T e process for preparing detoxicated proteins and bacterial vaccines which comprises treatingcapsulated bacteria and bacterial toxic proteins with alkali to break the capsules, treating with a digestant and then treating with a soap solution to separate the ca sules.

The process for preparing d'etoxicated proteins and bacterial vaccines which comprises treating capsulated bacteria and bacterial toxic proteins with alkali to break the capsules, treating with a digestant and then treating with sodium oleate to remove the capsules.

8. The process for preparing detoxicated proteins and bacterial vaccines which comprises treating capsulated bacteria and bacterial toxic proteins with alkali to break the capsules, treating with a digestant and then treating with a 15% solution of sodium oleate to remove the capsules.

9. The process for preparing detoxicated proteins and bacterial vaccines which comprises treating capsulated bacteria and bacterial toxic proteins with alkali to break the capsules, digesting with trypsin, treating with a soap solution to remove the capsules, treating with phenol, and centrifuging.

10. The process for preparing detoxicatecl proteins and bacterial vaccines which comprises treating capsulated bacteria and bacterial toxic proteins with alkali to break the capsules, treating with a digestant, treating with a soap solution to remove the capsules, treating with phenol, and centrifuging.

FRANK MONTGOMERY woon 

